Bead Based Assays for

Cytokine Detection

September 27, 2014

6th EFIS-EJI South East European

Immunology School – SEEIS 2014

Timisoara, Romania

The Cells of the Immune System

The Immune Reaction

(Th2)

(Th1)

The Immune Reaction

T cell

B cell

Mast cell

Neutrophil

Basophil

Eosinophil Macrophage

IL-2, 4, 6, 8, 9, IFNa, b, g

IL-1, 6, 8, 10, 12, 15,

IFNa, b, TNFa

IL-5

IL-4

IL-10

IL-1, 2, 4, 6, 10, 13,

14, IFNg, TNFa, b

IL-1, 3, 4, 10, 13,

IFNa, b, g, TNFa, b

IL-1, 2, 4, 6, 8, 9,

IFNa, b, g

IL-10, 12, TNFa, b

IL-3

, 4, 5

IL-4

IFNa, b, TNFa

IL-4

IL-1, IFNa, b, TNFa, b

IL-1, 6, 10, 12,

IFNa, b, TNFa, b

Applications for Monitoring the Immune

Functions

Tools & Technologies for Immune

Function Monitoring

Applications for Monitoring the Immune

Functions – Surface & Intracellular

Analysis by Flow Cytometry Block Fc Rceptors

&

Cell Surface Staining

Anti–IFN–g

CD

69

Principle of Cell Surface Staining

CD8

Principle of Intracellular Cytokine

Staining (ICS) Procedure

Fixation

&

Permeabilization

Analysis by Flow Cytometry

wash

Block Fc Rceptors

&

Cell Surface Staining

Intracellular Staining

Block Protein

Secretion

Stimulation

Anti–IFN–g

CD

69

1

2

3

4

5 6

7

1. Selected Stimulation Methods

A. TCR mediated (CD3/CD28)

B. Mitogenic (PHA/Con A)

C. Polyclonal (PMA/Ionomycin)

D. Lipopolysacharride (LPS)

E. Superantigen (SEB)

F. Antigen specific (Peptides)

Myeloid cell B cell T-helper cell Cytotox T cell

PMA/Iono Yes Yes Yes

PHA/ConA - - Very strong

LPS Yes/ better + IFNg Yes -

CD3/CD28 - - Yes/ better +IL-2

IL-2 - Yes Yes

Other cytokines Normally weak/very individual

-> not possible to make general assumptions

SEB +

CD28/CD49d - - Very strong

1. Selected Stimulation Methods Summary

1. Selected Stimulation Methods Conclusion

Artificial activation

• Easy to perform

• In most cases strong cellular reaction in ALL susceptible cells

• BUT: No direct link to specific and natural processes

• Very good positive control IN PARALLEL to natural activations

Natural / specific activation

• Often quite difficult to perform perfectly well

• Often only weak signals

• Often only in very small BUT VERY SPECIFIC subpopulations

• IN THE END the only relevant system working with primary cells

2. Protein Transport Inhibitors

GolgiPlug (Brefeldin A) vs. GolgiStop (Monensin)

2. Protein Transport Inhibitors

• Cytokines are secreted fast upon expression

• Cytokine concentrations in cells are very low / not

detectable

• Accumulation needed

• Substances are needed that delay cytokine secretion

– Brefeldin A: BD GolgiPlug (cat# 555029): 1mg / ml in DMSO (1mM)

– Monensin: BD GolgiStop (cat# 554724): 2mg / ml in EtOH (1mM)

Species Cytokines Transport

Inhibitor

Human IL-1a, IL-6, IL-8,

TNF-a Monensin

Human

IFN-g, IL-2, IL-10,

IL-12, MCP-1,

MCP-3, MIG,

MIP-1a, RANTES

Monensin or

Brefeldin A

Mouse IL-6, IL-12, TNF-a Brefeldin A

Mouse GM-CSF, IL-3,

IL-4, IL-5, IL-10 Monensin

Mouse IFN-g, IL-2 Monensin or

Brefeldin A

2. Protein Transport Inhibitors

Monensin (BD GolgiStop™) &

Brefeldin A (BD GolgiPlug™) are

commonly used to trap cytokines

inside the cell for analysis.

– Monensin prevents protein secretion

by interacting with the Golgi

transmembrane Na++/H+ transport.

– Brefeldin A redistributes intracellularly

produced proteins from the cis/medial

Golgi complex to the endoplasmic

reticulum.

– Different inhibitors may work better for

detection of different cytokines

2. Protein Transport Inhibitors Challenges

• Inhibition of intracellular protein transport by Monensin and

Brefeldin A is toxic for cells

• Monensin and Brefeldin A do not only delay secretion of

cytokines, but ALL protein secretion

• Maximum incubation time is 12-16hrs (protocol- and lab-

dependent)

• Not all cytokines are expressed at high levels directly after

activation

• Evaluate kinetics of cytokine expression for each activation

method

• Perform activation and add Brefeldin A or Monensin only for

the last 12hr

Effect of Protein Transport Inhibitors

No GolgiStop With GolgiStop

3. Blocking of Unspecific Staining

• Mouse and Rat Systems

– BD FcBlock™ (anti-CD32/CD64)

• Human Systems

– Excess of irrelevant purified Ig

– Human serum

4. Cell Surface Antigen Staining

5. Cell Fixation and Permeabilization

Fix prior to permeabilization to maintain structural

integrity

Fix = Paraformaldehyde

Permeabilize = Saponin

6. Controls for ICS

Purified Antibody Recombinant Cytokine Isotype Control

6. Controls for ICS

• Demonstrates inherent “non-specific” background

levels

• Negative staining control

• Subtract “non-specific” staining from positive

cytokine staining, these are actual positives

6. Controls for ICS Control Cells

• Fixed cytokine positive cells

– Staining control

– Workflow control

– Antibody titration

• Human

– HiCK-1, HiCK-2, HiCK-3, HiCK-4

• Mouse

– MiCK-1 (IL-2, IFN-g, TNF-a)

– MiCK-2 (IL-3, IL-4, IL-10, GM-CSF)

Detection & Quantitation of Soluble Proteins,

Transcription Factors & Phospho-Proteins ELISA

Principle of an ELISA

ELISA: Enzyme Linked Immunosorbent Assay

How does an ELISA work?

Principle of an ELISA

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

• Measures soluble proteins in supernatants and lysates

• Quantitative results

• Highly sensitive compared to single Ab applications

• Fast results

• No single cell analysis

ELISA Technology Features

Detection & Quantitation of Soluble Proteins,

Transcription Factors & Phospho-Proteins CBA

• Fluorescent immuno-assay allowing the simultaneous

detection and quantification of proteins within one

sample using flow cytometry

• CBA:

– Cytometric: Data acquired on a flow cytometer is analysed using

specific software

– Bead: The system is composed of various beads with different

fluorescence intensity coated with high affinity capture antibodies

– Array: Technology allowing multiplex analysis

CBA - Definition

Multiple

sizes Different

intensities

Different colors with

different intensities

Beads provide an expandable assay platform for use

with a flow cytometer

The Cytometric Bead Array (CBA)

CBA Kits CBA Flex Sets

Enhanced Sensitivity Flex Sets

How does a CBA Assay works ?

+

+

Analyte of Interest Fluorescent Detector Ab

Capture Bead Capture Ab

Acquire samples Prepare samples Analyze samples

IL-2

TNF

IFNg

IL-4

IL-10

IL-5

FCAP

software

CBA Basics

1 2 3

One Step

3 h incubation

Principle of BD™ CBA Kits

Wash buffer

Assay Buffer

Cytokine standard

Capture-Beads

Detection-antibodies

Instrument calibration

BD™ CBA Kits

Human Portfolio Kit content

Anaphylatoxin Kit C4a, C3a, C5a

Chemokine Kit IL-8, RANTES, MIG, MCP-1, IP-10

Inflammation Kit IL-8, IL-1b, IL-6, IL-10, TNF, IL-12p70

Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, IL-10, TNF, IFNg

Th1/Th2 Cytokine Kit II IL-2, IL-4, IL-6, IL-10, TNF, IFNg

Mouse Portfolio

Immunoglobulin Isotyping Kit IgG1, IgG2a, IgG2b, IgG3, IgA, IgM, IgE

Inflammation Kit IL-6, IL-10, MCP-1, IFNg, TNF, IL-12p70

Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, TNF, IFNg

Non-Human Primate

Th1/Th2 Cytokine Kit IL-2, IL-4, IL-5, IL-6, TNF, IFNg

Standards

Human Inflammation Standard IL-8, IL-1b, IL-6, IL-10, TNF, IL-12p70

Human Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, IL-6, IL-10, TNF, IFNg

Mouse Inflammation Standard IL-6, IL-10, MCP-1, IFNg, TNF, IL-12p70

Mouse Th1/Th2 Cytokine Standard IL-2, IL-4, IL-5, TNF, IFNg

BD™ CBA Kits

BD™ CBA Kits

• Preconfigured kits for consistent results with routine

panels

• Available for functional areas of biology such as

Th1, Th2, Th17, inflammatory cytokines,

anaphylatoxins

• Measure up to seven analytes simultaneously

• Compatible with flow cytometers that have a 488nm

laser

Total 2-3

hours

1 hour 1 or 2 hours

CBA Soluble

Protein Flex Set

CBA Cell Signaling

Flex Set (cell lysate)

Total 4 hours

3 hours 1 hour Beads

in bead diluent w/o colors

Beads + Sample/standard with assay diluent

yellow

Beads + Sample/standard +

detector with detector

diluent = green

Principle of BD CBA Flex Sets

CBA CBA Flex

No. of beads 6-7 30

Bead fluorescence FL-3 (670nm) or FL-4 (660nm) FL-3 (670 or 785)

FL-4 (660)

Detection method PE PE

Sample In solution

In solution

Cell lysate ; Cell Signaling

Flow cytometer 1 laser 488nm 2 lasers : 488 & 635

or 532 & 635

Software FCAP FCAP

CBA Kits vs. CBA Flex Sets

30 bead positions available

CBA Flex Sets: Bead Identification

A

4 5 6 7 8 9

B C D E

BD™ CBA Flex Sets

100 tests, packed as “set”

• Capture beads

• Detector reagents

• Standard (x2)

• Contains a simple TDS with performance info

BD™ CBA Flex Master Buffer Kit

100 and 500 test sizes

• includes all buffers and setup reagents

• Mouse/Rat

• Human

• Cell Signaling

• Contains a Manual with setup instructions and assay protocol details

BD CBA Master Buffer Kit

CBA Kits CBA Flex Sets

Signaling Flex Sets Soluble Protein Flex Sets Enhanced Sensitivity Flex Sets

contained

in the kit

Cell Signaling

Master Buffer Kit

(for 500 or 100

tests)

Human Soluble Protein

Master Buffer Kit (for 500

or 100 tests)

Mouse/Rat Soluble Protein

Master Buffer Kit ( for 500

or 100 tests)

Human Enhanced Sensitivity

Master Buffer Kit (for 500 or 100

tests)

Mouse Enhanced Sensitivity

Master Buffer Kit (for 500 or 100

tests)

The different types of flex sets cannot be mixed and performed in the same sample

BD™ CBA Flex Portfolio

• Soluble Proteins – Human (e.g. G-CSF, GM-CSF, IFNg, IL-1b, IL-2, IL-3,…)

• Soluble Proteins – Immunoglobulins (e.g. Total IgG, IgG1, IgG2,…)

• Phospho-specific CBA Flex Sets (e.g. Akt1, Akt2, Akt3, ERK1/2,…)

• Soluble Proteins – Mouse (e.g. IFNg, IL-1a, IL-2, IL-3, IL-4, IL-5,…)

• Soluble Proteins – Rat (e.g. IFNg, IL-4, IL-6, IL-10, TNF)

• Soluble Proteins – Supporting Reagents (e.g. Hu / Ms Master Buffer Kit,...)

• Soluble Proteins – Standards (e.g. IFNg, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-8,...)

• Functional Beads (positions A4 – E9, own specificities)

• Functional Beads – Supporting Reagents (buffer, IgG detector ab)

Total 2-3

hours

Capture

1 hour

Detection 1

or 2 hours

Classic Soluble

CBA Flex

Total 5 hours

Capture

2 hours

Detection

3 hours

Enhanced

Sensitivity CBA

Flex

Need to detect lower levels of analyte?

ES Flex Sets vs. Classical Flex Sets

BD CBA Enhanced Sensitivity Flex Sets

• A new family of Flex Sets

– Assay range:

• Allows cytokine detection in highly diluted samples

• Based on established CBA antibody pairs

• Tested in supernatant, serum, and plasma samples

– Main advantage in supernatant samples: can detect at earlier time

points - that were previously below the level of detection

– “Normal” serum and plasma samples still have undetectable

amounts of analyte

Classic CBA Flex Set Enhanced Sensitivity Flex Set

10 - 2500 pg/ml 0.274 – 200 pg/ml

BD CBA Enhanced Sensitivity Flex Sets

• Flex Set (100 Tests)

– One vial of capture beads

– One vial of detection reagent (Part A)

– Two vials of standard

• Master Buffer Kit (100 test and 500 test)

– Contains all buffers and set-up reagents

• No Bead Diluent for Serum/Plasma

• Contains more wash buffer

– Contains the Enhanced Sensitivity Detection Reagent (Part B)

Flex Set ES Flex Set Note: No data reported when falling below the last standard curve point

Human PBMC stimulated with PMA/Ionomycin

ES Flex Sets Can Detect Analytes Earlier

in a Time Course

Features and Benefits

Feature Benefits

Assay range of 274 – 200,000 fg/mL

Can detect early points in a time course and

highly diluted samples

35 fold sensitivity improvement as compared

to classic CBA

Each ES CBA flex set has a different bead

position

A fully flexible and cost-effective multiplex

panel as compared to kits that may include

inappropriate cytokines

Analyzes up to 10 soluble proteins with just

25 to 50 μL of sample

Based on existing Flex Set technology Fast and easy implementation

Acquisition of Samples

The BD Accuri C6 Flow Cytometer System

An affordable, full-featured, easy-to-use flow cytometer.

Two lasers and six detectors

Analysis of Data

• FCAP Array v3.0

Analysis Software

– Compatible with FCS 2.0 or

3.0 files from any flow

cytometer

– Integrated workflow for the

BD FACSVerse flow

cytometer

– Windows® 7, Vista®, or XP

compatible

Accuri C6 & CBA Summary

• A perfect and complete solution to measure & quantify

multiple proteins simultaneously

– CBA Flex Sets & Kits

• Broad spectrum of specificities, ready-to-use or full flexibility

– BD Accuri C6 with C6Sampler option

• Can be also used for other flow cytometric analysis

• Specific templates available for acquisition & analysis of CBA Kits and Flex Sets

– FCAP Array software

– Application and technical support

Feature of CBA Products

• Up to 30 proteins detectable in parallel

– Pre-configured kits or individual sets to mix and match your demands

• Sensitivity limit as low as 0.274 pg/mL

• Only 25 to 50 µL of sample needed

– Multiple quantitative results from a single small-volume sample

• Significantly reduced time to results

– High-performance optimized reagents for shortest time-to-results

– No assay formulation required, regardless of plex size

– Automated sample acquisition and increased throughput options with 96-well plate analysis

• A complete solution

– Broad instrument compatibility and dedicated analysis software

Applications for Monitoring the Immune

Functions